Project Summary Complex tissues such as the retina and brain rely on a vast array of specialized cell types to function properly. The diversity of cell types arises through development and maintenance of cell type-specific gene expression programs, driven by the activation of cis-regulatory elements (CREs; e.g., enhancer/promoters). Therefore, mapping the location and activity of CREs is integral in our understanding of cell type identity, predicting the impact of non-coding genetic variants in disease, and developing targeted therapies. In the retina, photoreceptor and bipolar cells are considered `sister' cell types, and offer an attractive model for comparing CREs in closely related cell types. While photoreceptor cells have been studied extensively, the chromatin landscape of bipolar cell types remains unknown. The objectives of this application are to elucidate the cis-regulatory architecture of bipolar cells in the wild type and remodeled mouse retina and to engineer enhancers for targeted expression in bipolar cells. Using transgenic marker lines, I have isolated ON, OFF, and aggregate bipolar cells from the mouse retina by FACs, and profiled the transcriptome and epigenome via mRNA seq and ATAC-seq, respectively. In Aim 1 I will correlate these datasets and compare data from photoreceptor and other cell types to identify potential CREs that define bipolar cells. The activity of potential CREs will be analyzed in vivo in individual cell types using CRE-seq, a massively parallel reporter assay delivered to the retina by AAV. These data will provide insight into the role of bipolar cells in processing the visual signal as well as their relationship to photoreceptor cells. In Aim 2 I will use a mouse model of retinal degeneration to identify the changes within the bipolar epigenome and transcriptome that occur during retinal remodeling. This information will guide therapeutic approaches targeting bipolar cells following the loss of photoreceptors. In Aim 3 I will identify CREs specific to ON and OFF bipolar cells. These elements will be subjected to saturating mutagenesis and further assayed for activity in order to engineer optimized enhancers for targeted expression. Together, these studies will provide a comprehensive map of CREs that define bipolar cell types. Additionally, they will generate constructs for specific, tunable expression in ON and OFF bipolar cells in both normal and degenerated retinas.